Current gold standard tissue diagnosis for renal allograft rejection is based on light microscopic evaluation of biopsies aided by a semi-quantitative scoring system combined with limited immunophenotypical and electron microscopic analysis. Major weaknesses of this system include poor inter-observer reproducibility, over- simplification of the pathogenetic processes, and applying arbitrary histologic criteria to define disease categories. Furthermore, in spite of the fact that a significnt proportion of the pathologic changes in the transplanted kidneys are linked to cellular and/or antibody-mediated anti-graft immunologic activity, no comprehensive tissue based assays are available to assess the cellular and molecular underpinnings of these processes. The emerging molecular diagnostic approaches such as gene expression profiling have their own shortcomings particularly in their lack of cellular and structural context and limited mechanistic insights of the disease process can be gleaned. Therefore, the potential diagnostic power of allograft kidney biopsies is currently under-utilized. In this proposal, we aim to 1) develop multiplex immunofluorescence and in situ hybridization (miFish) assays to characterize inflammation and structural alterations in the kidney allograft biopsies and 2) and validate the miFish assays against the current gold standard of Banff scoring and molecular profiling to better define disease categories. Overall, the miFish assays will aim to develop in this proposal will help to identify prognostic markers and therapeutic targets for kidney allograft pathologies. t may also be used to re-define the current diagnostic criteria for some of the problematic categories in Banff, such as borderline changes. Although the miFish assays we propose to develop will be optimized to a transplant environment in the kidneys, the very same assays should also be applicable (with some modifications) to assess inflammation in other settings, such as various inflammatory conditions in multiple organs, including autoimmune diseases, and also in neoplastic lesions.